Human requiem

ABSTRACT

Human REQUIEM polypeptides and DNA (RNA) encoding such REQUIEM and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such REQUIEM for the treatment of a susceptibility to viral infection, tumorogenesis and to diseases and defects in the control embryogenesis and tissue homeostasis, and the nucleic acid sequences described above may be employed in an assay for ascertaining such susceptibility. Antagonists against such REQUIEM and their use as a therapeutic to treat Alzheimer&#39;s disease, Parkinson&#39;s disease, rheumatoid arthritis, septic shock, sepsis, stroke, CNS inflammation, osteoporosis, ischemia reperfusion injury, cell death associated with cardiovascular disease, polycystic kidney disease, apoptosis of endothelial cells in cardiovascular disease, degenerative liver disease, MS, ALS, cererbellar degeneration, ischemic injury, myocardial infarction, AIDS, myelodysplastic syndromes, aplastic anemia, male pattern baldness, and head injury damage are also disclosed. Also disclosed are diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences and altered concentrations of the polypeptides. Also disclosed are diagnostic assays for detecting mutations in the polynucleotides encoding REQUIEM polypeptide and for detecting altered levels of the polypeptide in a host.

This application claims the benefit of U.S. Provisional Application No.60/021,229, filed Jun. 26, 1996.

This invention relates, in part, to newly identified polynucleotides andpolypeptides; variants and derivatives of the polynucleotides andpolypeptides; processes for making the polynucleotides and thepolypeptides, and their variants and derivatives; agonists andantagonists of the polypeptides; and uses of the polynucleotides,polypeptides, variants, derivatives, agonists and antagonists. Inparticular, in these and in other regards, the invention relates topolynucleotides and polypeptides of a human homologue of murine requiemgene, hereinafter referred to as "REQUIEM".

BACKGROUND OF THE INVENTION

Hematopoietic stem cell proliferation, differentiation and apoptosis,are regulated by various growth factors and cytokines (Metcalf, D.,Science 254, 529-531 (1992) and Vaux et al., Nature 335, 440-442(1988)). Such cells committed to the myeloid lineage express highaffinity receptors for GM-CSF1 and IL-3 (Miyajima et al., Blood82,1960-1973 (1993)). Moreover, such cells develop a requirement forGM-CSF or IL-3 to survive and undergo apoptosis in response to GM-CSF orIL-3 deprivation (Williams et al., Nature 343, 76-79 (1990)).

Hematopoietic cells are not unique in their requirement for growthfactors for survival. Throughout development, cells of all lineagesrequire proper signals to grow and differentiate. Failure to receivethese signals often results in death of the cells by apoptosis. Even inthe adult organism there is constant renewal and/or maintence of cellsthat requires growth factors and cytokines. Inappropriate cessation ofsignals from these growth factors may result in apoptosis therebycausing disfunction or disease.

Several apoptotic genes appear to be conserved in multicellularorganisms. For example, the ced-3 gene of Caenorhabditis elegans has afamily of mammalian homologue cysteine protease, includinginterleukin-1β-converting enzyme (ICE), Ich-1, CPP32, TX, ICEre1III, andLAP3 (Yuan, et al., Cell 75, 641-652 (1993) and Miura, et al., Cell 75,653-660 (1993); Wang, et al., Cell 78, 739-750 (1994),Fernandes-Alnemri, et al., JBC 269, 30761-30764 (1994), Faucheu, et al.,EMBO J 14, 1914-1922 (1995), Munday, et al., JBC 270, 15870-15876 (1995)and Duan, et al., JBC 271, 35013-35035 (1996)). Antagonists of theapoptosis are also conserved, such as, for example, ced-9 of C. elegansand its mammalian homologue bcl-2. Molecular control of initiation isless well resolved.

Cell lines that proliferate in response to IL-3 and undergo rapidprogrammed cell death following IL-3 deprivation can be used forexpression cloning systems. Using such a system, genes that antagonizeapoptosis may be found. Murine cells have been shown to be useful forthis purpose. A murine Requiem gene believed to encode a transcriptionfactor required for apoptosis response following survival factorwithdrawal from myeloid cells has been described (Gabig, et al., JBC269, 29515-29519 (1994)).

The present invention provides a human homolog of the Requiem gene andpolypeptides encoded therefrom (herein "REQUIEM"). The human sequencecontains more 5' sequence than the mouse requiem sequence and has a more5' start site which is believed to be the correct start site.

SUMMARY OF THE INVENTION

Toward these ends, and others, it is an object of the present inventionto provide polypeptides, inter alia, that have been identified as novelREQUIEM by homology between the amino acid sequence set out in FIG. 1and known amino acid sequences of other proteins such as those sequencesset out in FIG. 2.

It is a further object of the invention, moreover, to providepolynucleotides that encode REQUIEM, particularly polynucleotides thatencode the polypeptide herein designated REQUIEM.

In a particularly preferred embodiment of this aspect of the inventionthe polynucleotide comprises the region encoding human REQUIEM as setforth in FIG. 1.

In accordance with this aspect of the present invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressed by the human cDNA of FIG. 1 encoding the polypeptide in FIG.1.

In accordance with this aspect of the invention there are providedisolated nucleic acid molecules encoding human REQUIEM, including mRNAs,cDNAs, genomic DNAs and, in further embodiments of this aspect of theinvention, biologically, diagnostically, clinically or therapeuticallyuseful variants, analogs or derivatives thereof, or fragments thereof,including fragments of the variants, analogs and derivatives.

Among the particularly preferred embodiments of this aspect of theinvention are naturally occurring allelic variants of human REQUIEM.

It also is an object of the invention to provide REQUIEM polypeptides,particularly human REQUIEM polypeptides, that may be employed fortherapeutic purposes, for example, to treat viral infection, as ananti-tumor agent and to control embryonic development and tissuehomeostasis.

In accordance with this aspect of the invention there are provided novelpolypeptides of human origin referred to herein as REQUIEM as well asbiologically, diagnostically or therapeutically useful fragments,variants and derivatives thereof, variants and derivatives of thefragments, and analogs of the foregoing.

Among the particularly preferred embodiments of this aspect of theinvention are variants of human REQUIEM encoded by naturally occurringalleles of the human REQUIEM gene.

It is another object of the invention to provide a process for producingthe aforementioned polypeptides, polypeptide fragments, variants andderivatives, fragments of the variants and derivatives, and analogs ofthe foregoing.

In a preferred embodiment of this aspect of the invention there areprovided methods for producing the aforementioned REQUIEM polypeptidescomprising culturing host cells having expressibly incorporated thereinan exogenously-derived human REQUIEM-encoding polynucleotide underconditions for expression of human REQUIEM in the host and thenrecovering the expressed polypeptide. REQUIEM may also be purified fromnatural sources using any of many well known techniques.

In accordance with another object the invention there are providedproducts, compositions, processes and methods that utilize theaforementioned polypeptides and polynucleotides for research,biological, clinical, diagnostic and therapeutic purposes, inter alia.

In accordance with certain preferred embodiments of this aspect of theinvention, there are provided products, compositions and methods, interalia, for, among other things: assessing REQUIEM expression in cells bydetermining REQUIEM polypeptides or REQUIEM-encoding mRNA; as anantiviral agent, an anti-tumor agent and to control embryonicdevelopment and tissue homeostasis in vitro, ex vivo or in vivo byexposing cells to REQUIEM polypeptides or polynucleotides as disclosedherein; assaying genetic variation and aberrations, such as defects, inREQUIEM genes; and administering a REQUIEM polypeptide or polynucleotideto an organism to augment REQUIEM function or remediate REQUIEMdysfunction. Agonists targeted to defective cellular proliferation,including, for example, cancer cells and solid tumor cells may be usedfor the treatment of these diseses. Such targeting may be achieved viagene therapy of using antibody fusions. Agonists may also be used totreat follicular lymphomas, carcinomas associated with p53 mutations,autoimmune disorders, such as, for example, SLE, immune-mediatedglomerulonephritis; and hormone-dependent tumors, such as, for example,breast cancer, prostate cancer and ovary cancer; and viral infections,such as, for example, herpesviruses, poxviruses and adenoviruses.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided probes that hybridize tohuman REQUIEM sequences.

In certain additional preferred embodiments of this aspect of theinvention there are provided antibodies against REQUIEM polypeptides. Incertain particularly preferred embodiments in this regard, theantibodies are highly selective for human REQUIEM.

In accordance with another aspect of the present invention, there areprovided REQUIEM agonists. Among preferred agonists are molecules thatmimic REQUIEM, that bind to REQUIEM-binding molecules or receptormolecules, and that elicit or augment REQUIEM-induced responses. Alsoamong preferred agonists are molecules that interact with REQUIEM orREQUIEM polypeptides, or with other modulators of REQUIEM activitiesand/or gene expression, and thereby potentiate or augment an effect ofREQUIEM or more than one effect of REQUIEM.

In accordance with yet another aspect of the present invention, thereare provided REQUIEM antagonists. Among preferred antagonists are thosewhich mimic REQUIEM so as to bind to REQUIEM receptor or bindingmolecules but not elicit a REQUIEM-induced response or more than oneREQUIEM-induced response. Also among preferred antagonists are moleculesthat bind to or interact with REQUIEM so as to inhibit an effect ofREQUIEM or more than one effect of REQUIEM or which prevent expressionof REQUIEM.

In a further aspect of the invention there are provided compositionscomprising a REQUIEM polynucleotide or a REQUIEM polypeptide foradministration to cells in vitro, to cells ex vivo and to cells in vivo,or to a multicellular organism. In certain particularly preferredembodiments of this aspect of the invention, the compositions comprise aREQUIEM polynucleotide for expression of a REQUIEM polypeptide in a hostorganism for treatment of disease. Particularly preferred in this regardis expression in a human patient for treatment of a dysfunctionassociated with aberrant endogenous activity of REQUIEM.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill from the following description.It should be understood, however, that the following description and thespecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only. Various changes andmodifications within the spirit and scope of the disclosed inventionwill become readily apparent to those skilled in the art from readingthe following description and from reading the other parts of thepresent disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings depict certain embodiments of the invention. Theyare illustrative only and do not limit the invention otherwise disclosedherein.

FIG. 1 shows the nucleic acid and predicted amino acid sequence of humanREQUIEM (SEQ ID NOS: 1 and 2).

FIG. 2 shows a sequence alignment of human REQUIEM (top) and a knownmember of murine requiem (bottom).

GLOSSARY

The following illustrative explanations are provided to facilitateunderstanding of certain terms used frequently herein, particularly inthe examples. The explanations are provided as a convenience and are notlimitative of the invention.

DIGESTION of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes referred to herein are commerciallyavailable and their reaction conditions, cofactors and otherrequirements for use are known and routine to the skilled artisan.

For analytical purposes, typically, 1 μg of plasmid or DNA fragment isdigested with about 2 units of enzyme in about 20 μl of reaction buffer.For the purpose of isolating DNA fragments for plasmid construction,typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzymein proportionately larger volumes.

Appropriate buffers and substrate amounts for particular restrictionenzymes are described in standard laboratory manuals, such as thosereferenced below, and they are specified by commercial suppliers.

Incubation times of about 1 hour at 37° C. are ordinarily used, butconditions may vary in accordance with standard procedures, thesupplier's instructions and the particulars of the reaction. Afterdigestion, reactions may be analyzed, and fragments may be purified byelectrophoresis through an agarose or polyacrylamide gel, using wellknown methods that are routine for those skilled in the art.

GENETIC ELEMENT generally means a polynucleotide comprising a regionthat encodes a polypeptide or a region that regulates transcription ortranslation or other processes important to expression of thepolypeptide in a host cell, or a polynucleotide comprising both a regionthat encodes a polypeptide and a region operably linked thereto thatregulates expression.

Genetic elements may be comprised within a vector that replicates as anepisomal element; that is, as a molecule physically independent of thehost cell genome. They may be comprised within mini-chromosomes, such asthose that arise during amplification of transfected DNA by methotrexateselection in eukaryotic cells. Genetic elements also may be comprisedwithin a host cell genome; not in their natural state but, rather,following manipulation such as isolation, cloning and introduction intoa host cell in the form of purified DNA or in a vector, among others.

IDENTITY or SIMILARITY, as known in the art, are relationships betweentwo polypeptides as determined by comparing the amino acid sequence andits conserved amino acid substitutes of one polypeptide to the sequenceof a second polypeptide. Moreover, also known in the art is "identity"which means the degree of sequence relatedness between two polypeptideor two polynucleotides sequences as determined by the identity of thematch between two strings of such sequences. Both identity andsimilarity can be readily calculated (Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991). While there exist a number of methodsto measure identity and similarity between two polynucleotide orpolypeptide sequences, the terms "identity" and "similarity" are wellknown to skilled artisans (Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo,H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methodscommonly employed to determine identity or similarity between twosequences include, but are not limited to disclosed in Carillo, H., andLipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred methods todetermine identity are designed to give the largest match between thetwo sequences tested. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCG program package (Devereux, J., et al., Nucleic AcidsResearch 12(1): 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F. etal., J. Molec. Biol. 215: 403 (1990)).

ISOLATED means altered "by the hand of man" from its natural state;i.e., that, if it occurs in nature, it has been changed or removed fromits original environment, or both.

For example, a naturally occurring polynucleotide or a polypeptidenaturally present in a living animal in its natural state is not"isolated," but the same polynucleotide or polypeptide separated fromthe coexisting materials of its natural state is "isolated", as the termis employed herein. For example, with respect to polynucleotides, theterm isolated means that it is separated from the chromosome and cell inwhich it naturally occurs.

As part of or following isolation, such polynucleotides can be joined toother polynucleotides, such as DNAs, for mutagenesis, to form fusionproteins, and for propagation or expression in a host, for instance. Theisolated polynucleotides, alone or joined to other polynucleotides suchas vectors, can be introduced into host cells, in culture or in wholeorganisms. Introduced into host cells in culture or in whole organisms,such DNAs still would be isolated, as the term is used herein, becausethey would not be in their naturally occurring form or environment.Similarly, the polynucleotides and polypeptides may occur in acomposition, such as a media formulations, solutions for introduction ofpolynucleotides or polypeptides, for example, into cells, compositionsor solutions for chemical or enzymatic reactions, for instance, whichare not naturally occurring compositions, and, therein remain isolatedpolynucleotides or polypeptides within the meaning of that term as it isemployed herein.

LIGATION refers to the process of forming phosphodiester bonds betweentwo or more polynucleotides, which most often are double stranded DNAs.Techniques for ligation are well known to the art and protocols forligation are described in standard laboratory manuals and references,such as, for instance, Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989) ("Sambrook") and Maniatis et al., pg. 146, as citedbelow.

OLIGONUCLEOTIDES(S) refers to relatively short polynucleotides. Oftenthe term refers to single-stranded deoxyribonucleotides, but it canrefer as well to single- or double-stranded ribonucleotides, RNA:DNAhybrids and double-stranded DNAs, among others.

Oligonucleotides, such as single-stranded DNA probe oligonucleotides,often are synthesized by chemical methods, such as those implemented onautomated oligonucleotide synthesizers. However, oligonucleotides can bemade by a variety of other methods, including in vitro recombinantDNA-mediated techniques and by expression of DNAs in cells andorganisms.

Initially, chemically synthesized DNAs typically are obtained without a5' phosphate. The 5' ends of such oligonucleotides are not substratesfor phosphodiester bond formation by ligation reactions that employ DNAligases typically used to form recombinant DNA molecules. Where ligationof such oligonucleotides is desired, a phosphate can be added bystandard techniques, such as those that employ a kinase and ATP.

The 3' end of a chemically synthesized oligonucleotide generally has afree hydroxyl group and, in the presence of a ligase, such as T4 DNAligase, readily will form a phosphodiester bond with a 5' phosphate ofanother polynucleotide, such as another oligonucleotide. As is wellknown, this reaction can be prevented selectively, where desired, byremoving the 5' phosphates of the other polynucleotide(s) prior toligation.

PLASMIDS generally are designated herein by a lower case p precededand/or followed by capital letters and/or numbers, in accordance withstandard naming conventions that are familiar to those of skill in theart.

Starting plasmids disclosed herein are either commercially available,publicly available on an unrestricted basis, or can be constructed fromavailable plasmids by routine application of well known, publishedprocedures. Many plasmids and other cloning and expression vectors thatcan be used in accordance with the present invention are well known andreadily available to those of skill in the art. Moreover, those of skillreadily may construct any number of other plasmids suitable for use inthe invention. The properties, construction and use of such plasmids, aswell as other vectors, in the present invention will be readily apparentto those of skill from the present disclosure.

POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single-and double-stranded DNA, DNA that is a mixtureof single-and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions.

In addition, polynucleotide as used herein refers to triple-strandedregions comprising RNA or DNA or both RNA and DNA. The strands in suchregions may be from the same molecule or from different molecules. Theregions may include all of one or more of the molecules, but moretypically involve only a region of some of the molecules. One of themolecules of a triple-helical region often is an oligonucleotide.

As used herein, the term polynucleotide includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are"polynucleotides" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein.

It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells,inter alia.

POLYPEPTIDES, as used herein, includes all polypeptides as describedbelow. The basic structure of polypeptides is well known and has beendescribed in innumerable textbooks and other publications in the art. Inthis context, the term is used herein to refer to any peptide or proteincomprising two or more amino acids joined to each other in a linearchain by peptide bonds. As used herein, the term refers to both shortchains, which also commonly are referred to in the art as peptides,oligopeptides and oligomers, for example, and to longer chains, whichgenerally are referred to in the art as proteins, of which there aremany types.

It will be appreciated that polypeptides often contain amino acids otherthan the 20 amino acids commonly referred to as the 20 naturallyoccurring amino acids, and that many amino acids, including the terminalamino acids, may be modified in a given polypeptide, either by naturalprocesses, such as processing and other post-translationalmodifications, but also by chemical modification techniques which arewell known to the art. Even the common modifications that occurnaturally in polypeptides are too numerous to list exhaustively here,but they are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature, and they arewell known to those of skill in the art.

Among the known modifications which may be present in polypeptides ofthe present are, to name an illustrative few, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cystine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill and have beendescribed in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as, for instance PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as, for example,those provided by Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al., Analysis for protein modifications andnonprotein cofactors, Meth. Enzymol. 182: 626-646 (1990) and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62 (1992).

It will be appreciated, as is well known and as noted above, thatpolypeptides are not always entirely linear. For instance, polypeptidesmay be branched as a result of ubiquitination, and they may be circular,with or without branching, generally as a result of posttranslationevents, including natural processing event and events brought about byhuman manipulation which do not occur naturally. Circular, branched andbranched circular polypeptides may be synthesized by non-translationnatural process and by entirely synthetic methods, as well.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli, prior to proteolyticprocessing, almost invariably will be N-formylmethionine.

The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcell often carry out the same posttranslational glycosylations asmammalian cells and, for this reason, insect cell expression systemshave been developed to express efficiently mammalian proteins havingnative patterns of glycosylation, inter alia. Similar considerationsapply to other modifications.

It will be appreciated that the same type of modification may be presentin the same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.

In general, as used herein, the term polypeptide encompasses all suchmodifications, particularly those that are present in polypeptidessynthesized by expressing a polynucleotide in a host cell.

VARIANT(S) of polynucleotides or polypeptides, as the term is usedherein, are polynucleotides or polypeptides that differ from a referencepolynucleotide or polypeptide, respectively. Variants in this sense aredescribed below and elsewhere in the present disclosure in greaterdetail.

(1) A polynucleotide that differs in nucleotide sequence from another,reference polynucleotide. Generally, differences are limited so that thenucleotide sequences of the reference and the variant are closelysimilar overall and, in many regions, identical.

As noted below, changes in the nucleotide sequence of the variant may besilent. That is, they may not alter the amino acids encoded by thepolynucleotide. Where alterations are limited to silent changes of thistype a variant will encode a polypeptide with the same amino acidsequence as the reference. Also as noted below, changes in thenucleotide sequence of the variant may alter the amino acid sequence ofa polypeptide encoded by the reference polynucleotide. Such nucleotidechanges may result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence, as discussed below.

(2) A polypeptide that differs in amino acid sequence from another,reference polypeptide. Generally, differences are limited so that thesequences of the reference and the variant are closely similar overalland, in many regions, identical.

A variant and reference polypeptide may differ in amino acid sequence byone or more substitutions, additions, deletions, fusions andtruncations, which may be present in any combination.

RECEPTOR MOLECULE, as used herein, refers to molecules which bind orinteract specifically with REQUIEM polypeptides of the presentinvention, including not only classic receptors, which are preferred,but also other molecules that specifically bind to or interact withpolypeptides of the invention (which also may be referred to as "bindingmolecules" and "interaction molecules," respectively and as "REQUIEMbinding molecules" and "REQUIEM interaction molecules." Binding betweenpolypeptides of the invention and such molecules, including receptor orbinding or interaction molecules may be exclusive to polypeptides of theinvention, which is very highly preferred, or it may be highly specificfor polypeptides of the invention, which is highly preferred, or it maybe highly specific to a group of proteins that includes polypeptides ofthe invention, which is preferred, or it may be specific to severalgroups of proteins at least one of which includes polypeptides of theinvention.

Receptors also may be non-naturally occurring, such as antibodies andantibody-derived reagents that bind specifically to polypeptides of theinvention.

DESCRIPTION OF THE INVENTION

The present invention relates to novel REQUIEM polypeptides andpolynucleotides, among other things, as described in greater detailbelow. In particular, the invention relates to polypeptides andpolynucleotides of a novel human REQUIEM, which is related by amino acidsequence homology to murine requiem polypeptide. The invention relatesespecially to REQUIEM having the nucleotide and amino acid sequences setout in FIG. 1. It will be appreciated that the nucleotide and amino acidsequences set out in FIG. 1 were obtained by sequencing the cDNAobtained from a human cDNA library made from human cerebellum.

Polynucleotides

In accordance with one aspect of the present invention, there areprovided isolated polynucleotides which encode the REQUIEM polypeptidehaving the deduced amino acid sequence of FIG. 1.

In accordance with another aspect of the present invention, there areprovided isolated polynucleotides which encode the REQUIEM polypeptidehaving the deduced amino acid sequence of FIG. 1.

Using the information provided herein, such as the polynucleotideprimers sequences set out in Example 1, a polynucleotide of the presentinvention encoding human REQUIEM polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA from cells from microvascular endothelial tissue asstarting material. Illustrative of the invention, the polynucleotide ofthe invention was discovered as described in Example 1. REQIUEM can alsobe obtained from other tissues and cDNA libraries, for example,libraries derived from cells of human tissue and cell lines such as,endothelial cells and CEM cells.

Human REQUIEM of the invention is structurally related to other proteinsof the requiem family, as shown by the results of sequencing the cDNAencoding human REQUIEM in FIG. 1. The cDNA of derived using the primersset forth in Example 1 was obtained as described in Example 1. Thepolypeptide of FIG. 1 is a protein has a deduced molecular weight ofabout 45 kDa.

Polynucleotides of the present invention may be in the form of RNA, suchas mRNA, or in the form of DNA, including, for instance, cDNA andgenomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof. The DNA may be double-strandedor single-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

The coding sequence which encodes the polypeptide may be identical tothe coding sequence of the polynucleotide of FIG. 1. It also may be apolynucleotide with a different sequence, which, as a result of theredundancy (degeneracy) of the genetic code, encodes the polypeptide ofthe DNA of FIG. 1.

Polynucleotides of the present invention which encode the polypeptide ofFIG. 1 may include, but are not limited to the coding sequence for themature polypeptide, by itself; the coding sequence for the maturepolypeptide and additional coding sequences, such as those encoding aleader or secretory sequence, such as a pre-, or pro- or prepro- proteinsequence; the coding sequence of the mature polypeptide, with or withoutthe aforementioned additional coding sequences, together withadditional, non-coding sequences, including for example, but not limitedto introns and non-coding 5' and 3' sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing--including splicing and polyadenylation signals, forexample--ribosome binding and stability of mRNA; additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities. Also provided is mature REQUIEMprocessed from its precursor molecule, via autocatalysis or by otherenzymes, to produce two subunits, which form an active heterodimer (bothsubunits) or tetramer (two sets heterodimers). Thus, for instance, thepolypeptide may be fused to a marker sequence, such as a peptide, whichfacilitates purification of the fused polypeptide. In certain preferredembodiments of this aspect of the invention, the marker sequence is ahexa-histidine peptide, such as the tag provided in the pQE vector(Qiagen, Inc.), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The HA tag corresponds to an epitopederived of influenza hemagglutinin protein, which has been described byWilson et al., Cell 37: 767 (1984), for instance.

In accordance with the foregoing, the term "polynucleotide encoding apolypeptide" as used herein encompasses polynucleotides which include asequence encoding a polypeptide of the present invention, particularlythe human REQUIEM having the amino acid sequence set out in FIG. 1. Theterm encompasses polynucleotides that include a single continuous regionor discontinuous regions encoding the polypeptide (for example,interrupted by introns) together with additional regions, that also maycontain coding and/or non-coding sequences.

The present invention further relates to variants of the herein abovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1. A variant of the polynucleotide may be a naturally occurringvariant such as a naturally occurring allelic variant, or it may be avariant that is not known to occur naturally. Such non-naturallyoccurring variants of the polynucleotide may be made by mutagenesistechniques, including those applied to polynucleotides, cells ororganisms.

Among variants in this regard are variants that differ from theaforementioned polynucleotides by nucleotide substitutions, deletions oradditions. The substitutions, deletions or additions may involve one ormore nucleotides. The variants may be altered in coding or non-codingregions or both. Alterations in the coding regions may produceconservative or non-conservative amino acid substitutions, deletions oradditions.

Among the particularly preferred embodiments of the invention in thisregard are polynucleotides encoding polypeptides having the amino acidsequence of REQUIEM set out in FIG. 1; variants, analogs, derivativesand fragments thereof, and fragments of the variants, analogs andderivatives.

Further particularly preferred in this regard are polynucleotidesencoding REQUIEM variants, analogs, derivatives and fragments, andvariants, analogs and derivatives of the fragments, which have the aminoacid sequence of the REQUIEM polypeptide of FIG. 1 in which several, afew, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues aresubstituted, deleted or added, in any combination. Especially preferredamong these are substitutions, additions and deletions, which do notalter the properties and activities of the REQUIEM. Also especiallypreferred in this regard are conservative substitutions. Most highlypreferred are polynucleotides encoding polypeptides having the aminoacid sequence of FIG. 1, without substitutions.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical to a polynucleotide encoding the REQUIEMpolypeptide having the amino acid sequence set out in FIG. 1, andpolynucleotides which are complementary to such polynucleotides.Alternatively, most highly preferred are polynucleotides that comprise aregion that is at least 80% identical to a polynucleotide encoding theREQUIEM polypeptide of the human cDNA and polynucleotides complementarythereto. In this regard, polynucleotides at least 90% identical to thesame are particularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

Particularly preferred embodiments in this reset, moreover, arepolynucleotides which encode polypeptides which retain substantially thesame biological function or activity as the mature polypeptide encodedby the cDNA of FIG. 1.

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term "stringent conditions" means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for cDNA and genomic DNA toisolate full-length cDNAs and genomic clones encoding REQUIEM and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the human REQUIEM gene. Such probes generally willcomprise at least 15 bases. Preferably, such probes will have at least30 bases and may have at least 50 bases. Particularly preferred probeswill have at least 30 bases and will have 50 bases or less.

For example, the coding region of the REQUIEM gene may be isolated byscreening using the known DNA sequence to synthesize an oligonucleotideprobe. A labeled oligonucleotide having a sequence complementary to thatof a gene of the present invention is then used to screen a library ofhuman cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to human disease, as further discussed herein relatingto polynucleotide assays, inter alia.

The polynucleotides may encode a polypeptide which is the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature polypeptide (when the mature form has more thanone polypeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, may facilitateprotein trafficking, may prolong or shorten protein half-life or mayfacilitate manipulation of a protein for assay or production, amongother things. As generally is the case in situ, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the present invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences which are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Polypeptides

The present invention further relates to a human REQUIEM polypeptidewhich has the deduced amino acid sequence of FIG. 1.

The invention also relates to fragments, analogs and derivatives ofthese polypeptides. The terms "fragment," "derivative" and "analog" whenreferring to the polypeptide of FIG. 1, means a polypeptide whichretains essentially the same biological function or activity as suchpolypeptide. Thus, an analog includes a proprotein which can beactivated by cleavage of the proprotein portion to produce an activemature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide. Incertain preferred embodiments it is a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 may be(i) one in which one or more of the amino acid residues are substitutedwith a conserved or non-conserved amino acid residue (preferably aconserved amino acid residue) and such substituted amino acid residuemay or may not be one encoded by the genetic code, or (ii) one in whichone or more of the amino acid residues includes a substituent group, or(iii) one in which the mature polypeptide is fused with anothercompound, such as a compound to increase the half-life of thepolypeptide (for example, polyethylene glycol), or (iv) one in which theadditional amino acids are fused to the mature polypeptide, such as aleader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

Among the particularly preferred embodiments of the invention in thisregard are polypeptides having the amino acid sequence of REQUIEM setout in FIG. 1, variants, analogs, derivatives and fragments thereof, andvariants, analogs and derivatives of the fragments. Alternatively,particularly preferred embodiments of the invention in this regard arepolypeptides having the amino acid sequence of the REQUIEM, variants,analogs, derivatives and fragments thereof, and variants, analogs andderivatives of the fragments.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Further particularly preferred in this regard are variants, analogs,derivatives and fragments, and variants, analogs and derivatives of thefragments, having the amino acid sequence of the REQUIEM polypeptide ofFIG. 1, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or noamino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are substitutions,additions and deletions, which do not alter the properties andactivities of the REQUIEM. Also especially preferred in this regard areconservative substitutions. Most highly preferred are polypeptideshaving the amino acid sequence of FIG. 1 without substitutions.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The polypeptides of the present invention include the polypeptide ofFIG. 1 (in particular the mature polypeptide) as well as polypeptideswhich have at least 80% identity to the polypeptide of FIG. 1 and morepreferably at least 90% similarity (more preferably at least 90%identity) to the polypeptide of FIG. 1 and still more preferably atleast 95% similarity (still more preferably at least 95% identity) tothe polypeptide of FIG. 1 and also include portions of such polypeptideswith such portion of the polypeptide generally containing at least 30amino acids and more preferably at least 50 amino acids.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

Fragments

Also among preferred embodiments of this aspect of the present inventionare polypeptides comprising fragments of REQUIEM, most particularlyfragments of the REQUIEM having the amino acid set out in FIG. 1, andfragments of variants and derivatives of the REQUIEM of FIG. 1.

In this regard a fragment is a polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the aforementioned REQUIEM polypeptides and variants or derivativesthereof.

Such fragments may be "free-standing," i.e., not part of or fused toother amino acids or polypeptides, or they may be comprised within alarger polypeptide of which they form a part or region. When comprisedwithin a larger polypeptide, the presently discussed fragments mostpreferably form a single continuous region. However, several fragmentsmay be comprised within a single larger polypeptide. For instance,certain preferred embodiments relate to a fragment of a REQUIEMpolypeptide of the present comprised within a precursor polypeptidedesigned for expression in a host and having heterologous pre andpro-polypeptide regions fused to the amino terminus of the REQUIEMfragment and an additional region fused to the carboxyl terminus of thefragment. Therefore, fragments in one aspect of the meaning intendedherein, refers to the portion or portions of a fusion polypeptide orfusion protein derived from REQUIEM.

As representative examples of polypeptide fragments of the invention,there may be mentioned those which have from about 5-15, 10-20, 15-40,30-55, 41-65, 41-80, 41-90, 50-100, 75-100, 90-115, 100-125, and 110-113amino acids long.

In this context about includes the particularly recited range and rangeslarger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid ateither extreme or at both extremes. For instance, about 40-90 aminoacids in this context means a polypeptide fragment of 40 plus or minusseveral, a few, 5, 4, 3, 2 or 1 amino acids to 90 plus or minus severala few, 5, 4, 3, 2 or 1 amino acid residues, i.e., ranges as broad as 40minus several amino acids to 90 plus several amino acids to as narrow as40 plus several amino acids to 90 minus several amino acids.

Highly preferred in this regard are the recited ranges plus or minus asmany as 5 amino acids at either or at both extremes. Particularly highlypreferred are the recited ranges plus or minus as many as 3 amino acidsat either or at both the recited extremes. Especially particularlyhighly preferred are ranges plus or minus 1 amino acid at either or atboth extremes or the recited ranges with no additions or deletions. Mosthighly preferred of all in this regard are fragments from about 5-15,10-20, 15-40, 30-55, 41-65, 41-80, 41-90, 50-100, 75-100, 90-115,100-125, and 110-113 amino acids long.

Among especially preferred fragments of the invention are truncationmutants of REQUIEM. Truncation mutants include REQUIEM polypeptideshaving the amino acid sequence of FIG. 1, or of variants or derivativesthereof, except for deletion of a continuous series of residues (thatis, a continuous region, part or portion) that includes the aminoterminus, or a continuous series of residues that includes the carboxylterminus or, as in double truncation mutants, deletion of two continuousseries of residues, one including the amino terminus and one includingthe carboxyl terminus. Fragments having the size ranges set out aboutalso are preferred embodiments of truncation fragments, which areespecially preferred among fragments generally.

Also preferred in this aspect of the invention are fragmentscharacterized by structural or functional attributes of REQUIEM.Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions("alpha-regions"), beta-sheet and beta-sheet-forming regions("beta-regions"), turn and turn-forming regions ("turn-regions"), coiland coil-forming regions ("coil-regions"), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions of REQUIEM.

Among highly preferred fragments in this regard are those that compriseregions of REQUIEM that combine several structural features, such asseveral of the features set out above. In this regard, the regionsdefined by the residues about 10 to about 20, about 40 to about 50,about 70 to about 90 and about 100 to about 113 of FIG. 1, which all arecharacterized by amino acid compositions highly characteristic ofturn-regions, hydrophilic regions, flexible-regions, surface-formingregions, and high antigenic index-regions, are especially highlypreferred regions. Such regions may be comprised within a largerpolypeptide or may be by themselves a preferred fragment of the presentinvention, as discussed above. It will be appreciated that the term"about" as used in this paragraph has the meaning set out aboveregarding fragments in general.

Further preferred regions are those that mediate activities of REQUIEM.Most highly preferred in this regard are fragments that have a chemical,biological or other activity of REQUIEM, including those with a similaractivity or an improved activity, or with a decreased undesirableactivity. Highly preferred in this regard are fragments that containregions that are homologs in sequence, or in position, or in bothsequence and to active regions of related polypeptides, such as therelated polypeptides set out in FIG. 2, which includes murine requiempolypeptide. Among particularly preferred fragments in these regards aretruncation mutants, as discussed above.

It will be appreciated that the invention also relates to, among others,polynucleotides encoding the aforementioned fragments, polynucleotidesthat hybridize to polynucleotides encoding the fragments, particularlythose that hybridize under stringent conditions, and polynucleotides,such as PCR primers, for amplifying polynucleotides that encode thefragments. In these regards, preferred polynucleotides are those thatcorrespondent to the preferred fragments, as discussed above. Preferredpolynucleotides fragments may be derived from the sequences of FIG. 1.

Vectors, host cells, expression

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells can be genetically engineered to incorporate polynucleotidesand express polypeptides of the present invention. For instance,polynucleotides may be introduced into host cells using well knowntechniques of infection, transduction, transfection, transvection andtransformation. The polynucleotides may be introduced alone or withother polynucleotides. Such other polynucleotides may be introducedindependently, co-introduced or introduced joined to the polynucleotidesof the invention.

Thus, for instance, polynucleotides of the invention may be transfectedinto host cells with another, separate, polynucleotide encoding aselectable marker, using standard techniques for co-transfection andselection in, for instance, mammalian cells. In this case thepolynucleotides generally will be stably incorporated into the host cellgenome.

Alternatively, the polynucleotides may be joined to a vector containinga selectable marker for propagation in a host. The vector construct maybe introduced into host cells by the aforementioned techniques.Generally, a plasmid vector is introduced as DNA in a precipitate, suchas a calcium phosphate precipitate, or in a complex with a chargedlipid. Electroporation also may be used to introduce polynucleotidesinto a host. If the vector is a virus, it may be packaged in vitro orintroduced into a packaging cell and the packaged virus may betransduced into cells. A wide variety of techniques suitable for makingpolynucleotides and for introducing polynucleotides into cells inaccordance with this aspect of the invention are well known and routineto those of skill in the art. Such techniques are reviewed at length inSambrook et al. cited above, which is illustrative of the manylaboratory manuals that detail these techniques.

In accordance with this aspect of the invention the vector may be, forexample, a plasmid vector, a single or double-stranded phage vector, asingle or double-stranded RNA or DNA viral vector. Such vectors may beintroduced into cells as polynucleotides, preferably DNA, by well knowntechniques for introducing DNA and RNA into cells. The vectors, in thecase of phage and viral vectors also may be and preferably areintroduced into cells as packaged or encapsidated virus by well knowntechniques for infection and transduction. Viral vectors may bereplication competent or replication defective. In the latter case viralpropagation generally will occur only in complementing host cells.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression. Such specific expression may be inducibleexpression or expression only in certain types of cells or bothinducible and cell-specific. Particularly preferred among induciblevectors are vectors that can be induced for expression by environmentalfactors that are easy to manipulate, such as temperature and nutrientadditives. A variety of vectors suitable to this aspect of theinvention, including constitutive and inducible expression vectors foruse in prokaryotic and eukaryotic hosts, are well known and employedroutinely by those of skill in the art.

The engineered host cells can be cultured in conventional nutrientmedia, which may be modified as appropriate for, inter alia, activatingpromoters, selecting transformants or amplifying genes. Cultureconditions, such as temperature, pH and the like, previously used withthe host cell selected for expression generally will be suitable forexpression of polypeptides of the present invention as will be apparentto those of skill in the art.

A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include chromosomal, episomaland virus-derived vectors e.g., vectors derived from bacterial plasmids,from bacteriophage, from yeast episomes, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such assimian virus 40 ("SV40"), vaccinia viruses, adenoviruses, fowl poxviruses, pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids, all maybe used for expression in accordance with this aspect of the presentinvention. Generally, any vector suitable to maintain, propagate orexpress polynucleotides to express a polypeptide in a host may be usedfor expression in this regard.

The appropriate DNA sequence may be inserted into the vector by any of avariety of well-known and routine techniques. In general, a DNA sequencefor expression is joined to an expression vector by cleaving the DNAsequence and the expression vector with one or more restrictionendonucleases and then joining the restriction fragments together usingT4 DNA ligase. Procedures for restriction and ligation that can be usedto this end are well known and routine to those of skill. Suitableprocedures in this regard, and for constructing expression vectors usingalternative techniques, which also are well known and routine to thoseskill, are set forth in great detail in Sambrook et al. cited elsewhereherein.

The DNA sequence in the expression vector is operatively linked toappropriate expression control sequence(s), including, for instance, apromoter to direct mRNA transcription. Representatives of such promotersinclude the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retoviralLTRs, to name just a few of the well-known promoters. It will beunderstood that numerous promoters not mentioned are suitable for use inthis aspect of the invention are well known and readily may be employedby those of skill in the manner illustrated by the discussion and theexamples herein.

In general, expression constructs will contain sites for transcriptioninitiation and termination, and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will include a translationinitiating AUG at the beginning and a termination codon appropriatelypositioned at the end of the polypeptide to be translated.

In addition, the constructs may contain control regions that regulate aswell as engender expression. Generally, in accordance with many commonlypracticed procedures, such regions will operate by controllingtranscription, such as repressor binding sites and enhancers, amongothers.

Vectors for propagation and expression generally will include selectablemarkers. Such markers also may be suitable for amplification or thevectors may contain additional markers for this purpose. In this regard,the expression vectors preferably contain one or more selectable markergenes to provide a phenotypic trait for selection of transformed hostcells. Preferred markers include dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, and tetracycline or ampicillinresistance genes for culturing E. coli and other bacteria.

The vector containing the appropriate DNA sequence as describedelsewhere herein, as well as an appropriate promoter, and otherappropriate control sequences, may be introduced into an appropriatehost using a variety of well known techniques suitable to expressiontherein of a desired polypeptide. Representative examples of appropriatehosts include bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Hosts for a greatvariety of expression constructs are well known, and those of skill willbe enabled by the present disclosure readily to select a host forexpressing a polypeptides in accordance with this aspect of the presentinvention.

More particularly, the present invention also includes recombinantconstructs, such as expression constructs, comprising one or more of thesequences described above. The constructs comprise a vector, such as aplasmid or viral vector, into which such a sequence of the invention hasbeen inserted. The sequence may be inserted in a forward or reverseorientation. In certain preferred embodiments in this regard, theconstruct further comprises regulatory sequences, including, forexample, a promoter, operably linked to the sequence. Large numbers ofsuitable vectors and promoters are known to those of skill in the art,and there are many commercially available vectors suitable for use inthe present invention.

The following vectors, which are commercially available, are provided byway of example. Among vectors preferred for use in bacteria are pQE70,pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO,pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,pMSG and pSVL available from Pharmacia. These vectors are listed solelyby way of illustration of the many commercially available and well knownvectors that are available to those of skill in the art for use inaccordance with this aspect of the present invention. It will beappreciated that any other plasmid or vector suitable for, for example,introduction, maintenance, propagation or expression of a polynucleotideor polypeptide of the invention in a host may be used in this aspect ofthe invention.

Promoter regions can be selected from any desired gene using vectorsthat contain a reporter transcription unit lacking a promoter region,such as a chloramphenicol acetyl transferase ("CAT") transcription unit,downstream of restriction site or sites for introducing a candidatepromoter fragment; i.e., a fragment that may contain a promoter. As iswell known, introduction into the vector of a promoter-containingfragment at the restriction site upstream of the cat gene engendersproduction of CAT activity, which can be detected by standard CATassays. Vectors suitable to this end are well known and readilyavailable. Two such vectors are pKK232-8 and pCM7. Thus, promoters forexpression of polynucleotides of the present invention include not onlywell known and readily available promoters, but also promoters thatreadily may be obtained by the foregoing technique, using a reportergene.

Among known bacterial promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli lacI and lacZ promoters, the T3 and T7promoters, the gpt promoter, the lambda PR, PL promoters and the trppromoter.

Among known eukaryotic promoters suitable in this regard are thecytomegalovirus ("CMV") immediate early promoter, the HSV thymidinekinase promoter, the early and late SV40 promoters, the promoters ofretroviral LTRs, such as those of the Rous sarcoma virus ("RoSV"), andmetallothionein promoters, such as the mouse metallothionein-I promoter.

Selection of appropriate vectors and promoters for expression in a hostcell is a well known procedure and the requisite techniques forexpression vector construction, introduction of the vector into the hostand expression in the host are routine skills in the art.

The present invention also relates to host cells containing theabove-described constructs discussed above. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al. BASIC METHODS IN MOLECULARBIOLOGY, (1986).

Constructs in host cells can be used in a conventional manner to producethe gene product encoded by the recombinant sequence. Alternatively, thepolypeptides of the invention can be synthetically produced byconventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook et al., cited above.

Generally, recombinant expression vectors will include origins ofreplication, a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, and a selectablemarker to permit isolation of vector containing cells after exposure tothe vector. Among suitable promoters are those derived from the genesthat encode glycolytic enzymes such as 3-phosphoglycerate kinase("PGK"), a-factor, acid phosphatase, and heat shock proteins, amongothers. Selectable markers include the ampicillin resistance gene of E.coli and the trp1 gene of S. cerevisiae.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Polynucleotides of the invention, encoding the heterologous structuralsequence of a polypeptide of the invention generally will be insertedinto the vector using standard techniques so that it is operably linkedto the promoter for expression. The polynucleotide will be positioned sothat the transcription start site is located appropriately 5' to aribosome binding site. The ribosome binding site will be 5' to the AUGthat initiates translation of the polypeptide to be expressed.Generally, there will be no other open reading frames that begin with aninitiation codon, usually AUG, and lie between the ribosome binding siteand the initiating AUG. Also, generally, there will be a translationstop codon at the end of the polypeptide and there will be apolyadenylation signal and a transcription termination signalappropriately disposed at the 3' end of the transcribed region.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, a region may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art.

Suitable prokaryotic hosts for propagation, maintenance or expression ofpolynucleotides and polypeptides in accordance with the inventioninclude Escherichia coli, Bacillus subtilis and Salmonella typhimurium.Various species of Pseudomonas, Streptomyces, and Staphylococcus aresuitable hosts in this regard. Moreover, many other hosts also known tothose of skill may be employed in this regard.

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,U.S.A.). These pBR322 "backbone" sections are combined with anappropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, where the selected promoteris inducible it is induced by appropriate means (e.g., temperature shiftor exposure to chemical inducer) and cells are cultured for anadditional period.

Cells typically then are harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can be employed for expression,as well. Examples of mammalian expression systems include the COS-6lines of monkey kidney fibroblast, described in Gluzman et al., Cell 23:175 (1981). Other cell lines capable of expressing a compatible vectorinclude for example, the C127, 3T3, CHO, HeLa, human kidney 293 and BHKcell lines.

Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation sites, splice donor and acceptor sites,transcriptional termination sequences, and 5' flanking non-transcribedsequences that are necessary for expression. In certain preferredembodiments in this regard DNA sequences derived from the SV40 splicesites, and the SV40 polyadenylation sites are used for requirednon-transcribed genetic elements of these types.

The REQUIEM polypeptide can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography ("HPLC") is employed for purification. Well knowntechniques for refolding protein may be employed to regenerate activeconformation when the polypeptide is denatured during isolation and orpurification.

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

REQUIEM polynucleotides and polypeptides may be used in accordance withthe present invention for a variety of applications, particularly thosethat make use of the chemical and biological properties of REQUIEM.Additional applications relate to diagnosis and to treatment ofdisorders of cells, tissues and organisms. These aspects of theinvention are illustrated further by the following discussion.

Polynucleotide assays

This invention is also related to the use of the REQUIEM polynucleotidesto detect complementary polynucleotides such as, for example, as adiagnostic reagent. Detection of a mutated form of REQUIEM associatedwith a dysfunction will provide a diagnostic tool that can add or definea diagnosis of a disease or susceptibility to a disease which resultsfrom under-expression over-expression or altered expression of REQUIEM.Individuals carrying mutations in the human REQUIEM gene may be detectedat the DNA level by a variety of techniques. Nucleic acids for diagnosismay be obtained from a patient's cells, such as from blood, urine,saliva, tissue biopsy and autopsy material. The genomic DNA may be useddirectly for detection or may be amplified enzymatically by using PCRprior to analysis. PCR (Saiki et al., Nature, 324: 163-166 (1986)). RNAor cDNA may also be used in the same ways. As an example, PCR primerscomplementary to the nucleic acid encoding REQUIEM can be used toidentify and analyze REQUIEM expression and mutations. For example,deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to radiolabeled REQUIEMRNA or alternatively, radiolabeled REQUIEM antisense DNA sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase A digestion or by differences in melting temperatures.

Sequence differences between a reference gene and genes having mutationsalso may be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230: 1242 (1985)).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,restriction fragment length polymorphisms ("RFLP") and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations also can be detected by in situ analysis.

In accordance with a further aspect of the invention, there is provideda process for determining disease associated with viral infection,tumorogenesis and to control embryogenesis and tissue homeostasis.Diseases associated with viral infection, tumorogenesis and to controlembryogenesis and tissue homeostasis, or a susceptibility to viralinfection, tumorogenesis and to diseases and defects in the control ofcontrol of embryogenesis and tissue homeostasis. Thus, a mutation inREQUIEM indicates a susceptibility to viral infection, tumorogenesis andto diseases and defects in the control embryogenesis and tissuehomeostasis, and the nucleic acid sequences described above may beemployed in an assay for ascertaining such susceptibility. Thus, forexample, the assay may be employed to determine a mutation in a humanREQUIEM protein as herein described, such as a deletion, truncation,insertion, frame shift, etc., with such mutation being indicative of asusceptibility to viral infection, tumorogenesis and to diseases anddefects in the control of embryogenesis and tissue homeostasis.

A mutation may be ascertained for example, by a DNA sequencing assay.Tissue samples, including but not limited to blood samples are obtainedfrom a human patient. The samples are processed by methods known in theart to capture the RNA. First strand cDNA is synthesized from the RNAsamples by adding an oligonucleotide primer consisting of polythymidineresidues which hybridize to the polyadenosine stretch present on themRNA's. Reverse transcriptase and deoxynucleotides are added to allowsynthesis of the first strand cDNA. Primer sequences are synthesizedbased on the DNA sequence of the REQUIEM protein of the invention. Theprimer sequence is generally comprised of at least 15 consecutive bases,and may contain at least 30 or even 50 consecutive bases.

Individuals carrying mutations in the gene of the present invention mayalso be detected at the DNA level by a variety of techniques. Nucleicacids for diagnosis may be obtained from a patient's cells, includingbut not limited to blood, urine, saliva, tissue biopsy and autopsymaterial. The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR (Saiki et al., Nature, 324: 163-166(1986)) prior to analysis. RT-PCR can also be used to detect mutations.It is particularly preferred to used RT-PCR in conjunction withautomated detection systems, such as, for example, GeneScan. RNA or cDNAmay also be used for the same purpose, PCR or RT-PCR. As an example, PCRprimers complementary to the nucleic acid encoding REQUIEM can be usedto identify and analyze mutations. For example, deletions and insertionscan be detected by a change in size of the amplified product incomparison to the normal genotype. Point mutations can be identified byhybridizing amplified DNA to radiolabeled RNA or alternatively,radiolabeled antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Primers, selected by well known methods, may be used for amplifyingREQUIEM cDNA isolated from a sample derived from a patient. Theinvention also provides the primers selected with 1, 2, 3 or 4nucleotides removed from the 5' and/or the 3' end. The primers may beused to amplify the gene isolated from the patient such that the genemay then be subject to various techniques for elucidation of the DNAsequence. In this way, mutations in the DNA sequence may be diagnosed.

Sequence differences between the reference gene and genes havingmutations may be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments may be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer isused with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230: 1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85: 4397-4401 (1985)).

Thus, the detection of a specific DNA sequence and/or quantitation ofthe level of the sequence may be achieved by methods such ashybridization, RNase protection, chemical cleavage, direct DNAsequencing or the use of restriction enzymes, (e.g., RestrictionFragment Length Polymorphisms (RFLP)) and Southern blotting of genomicDNA. The invention provides a process for diagnosing or detecting,disease, particularly Alzheimer's disease, Parkinson's disease,rheumatoid arthritis, septic shock, sepsis, stroke, CNS inflammation,osteoporosis, ischemia reperfusion injury, cell death associated withcardiovascular disease, polycystic kidney disease, apoptosis ofendothelial cells in cardiovascular disease, degenerative liver disease,MS, ALS, cererbellar degeneration, ischemic injury, myocardialinfarction, AIDS, myelodysplastic syndromes, aplastic anemia, malepattern baldness, and head injury damage, as well as a susceptibility toviral infection and cancer, an to detect aberrant control of embryonicdevelopment and tissue homeostasis, comprising determining from a samplederived from a patient altered expression of polynucleotide having thesequence of FIG. 1 as compared to normal control samples. Expression ofpolynucleotide can be measured using any on of the methods well known inthe art for the quantation of polynucleotides, such as, for example,PCR, RT-PCR, RNase protection, Northern blotting and other hybridizationmethods.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomallocation.

As an example of how this is performed, REQUIEM DNA is digested andpurified with QIAEX II DNA purification kit (QIAGEN, Inc., Chatsworth,Calif.) and ligated to Super Cos1 cosmid vector (STRATAGENE, La Jolla,Calif.). DNA is purified using Qiagen Plasmid Purification Kit (QIAGENInc., Chatsworth, Calif.) and 1 mg is labeled by nick translation in thepresence of Biotin-dATP using BioNick Labeling Kit (GibcoBRL, LifeTechnologies Inc., Gaithersburg, Md.). Biotinilation is detected withGENE-TECT Detection System (CLONTECH Laboratories, Inc. Palo Alto,Calif.). In situ Hybridization is performed on slides using ONCOR LightHybridization Kit (ONCOR, Gaithersberg, Md.) to detect single copysequences on metaphase chromosomes. Peripheral blood of normal donors iscultured for three days in RPMI 1640 supplemented with 20% FCS, 3% PHAand penicillin/streptomycin, synchronized with 10⁻⁶ M methotrexate for17 hours and washed twice with unsupplemented RPMI. Cells are incubatedwith 10⁻³ M thymidine for 7 hours. The cells are arrested in metaphaseafter 20 minutes incubation with colcemid (0.5 μg/ml) followed byhypotonic lysis in 75 mM KCl for 15 minutes at 37° C. Cell pellets arethen spun out and fixed in Carnoy's fixative (3:1 methanol/acetic acid).

Metaphase spreads are prepared by adding a drop of the suspension ontoslides and air dried. Hybridization is performed by adding 100 ng ofprobe suspended in 10 ml of hybridization mix (50% formamide, 2×SSC, 1%dextran sulfate) with blocking human placental DNA 1 μg/ml), Probemixture is denatured for 10 minutes in 70° C. water bath and incubatedfor 1 hour at 37° C., before placing on a prewarmed (37° C.) slide,which is previously denatured in 70% formamide/2×SSC at 70° C., anddehydrated in ethanol series, chilled to 4° C.

Slides are incubated for 16 hours at 37° C. in a humidified chamber.Slides are washed in 50% formamide/2×SSC for 10 minutes at 41° C. and2×SSC for 7 minutes at 37° C. Hybridization probe is detected byincubation of the slides with FITC-Avidin (ONCOR, Gaithersberg, Md.),according to the manufacturer protocol. Chromosomes are counterstainedwith propridium iodine suspended in mounting medium. Slides arevisualized using a Leitz ORTHOPLAN 2-epifluorescence microscope and fivecomputer images are taken using Imagenetics Computer and MacIntoshprinter.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (publicly available on line via computer).The relationship between genes and diseases that have been mapped to thesame chromosomal region are then identified through linkage analysisusing well known methods.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

Chromosome assays

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a REQUIEM gene. This can beaccomplished using a variety of well known techniques and libraries,which generally are available commercially. The genomic DNA is used forin situ chromosome mapping using well known techniques for this purpose.Typically, in accordance with routine procedures for chromosome mapping,some trial and error may be necessary to identify a genomic probe thatgives a good in situ hybridization signal.

In some cases, in addition, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp) from the cDNA. Computeranalysis of the 3' untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization ("FISH") of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60. For a review of this technique, see Verma et al.,HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, NewYork (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,MENDELIAN INHERITANCE IN MAN (publicly available on line via computer).The relationship between genes and diseases that have been mapped to thesame chromosomal region are then identified through linkage analysis(coinheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Polypeptide assays

The present invention also relates to a diagnostic assays such asquantitative and diagnostic assays for detecting levels of REQUIEMprotein in cells and tissues, including determination of normal andabnormal levels. Thus, for instance, a diagnostic assay in accordancewith the invention for detecting over-expression of REQUIEM proteincompared to normal control tissue samples may be used to detect thepresence of a tumor, for example. Assay techniques that can be used todetermine levels of a protein, such as an REQUIEM protein of the presentinvention, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.Among these ELISAs frequently are preferred. An ELISA assay initiallycomprises preparing an antibody specific to REQUIEM, preferably amonoclonal antibody. In addition a reporter antibody generally isprepared which binds to the monoclonal antibody. The reporter antibodyis attached a detectable reagent such as radioactive, fluorescent orenzymatic reagent, in this example horseradish peroxidase enzyme.

To carry out an ELISA a sample is removed from a host and incubated on asolid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein such as bovine serum albumin.Next, the monoclonal antibody is incubated in the dish during which timethe monoclonal antibodies attach to any REQUIEM proteins attached to thepolystyrene dish. Unbound monoclonal antibody is washed out with buffer.The reporter antibody linked to horseradish peroxidase is placed in thedish resulting in binding of the reporter antibody to any monoclonalantibody bound to REQUIEM. Unattached reporter antibody is then washedout. Reagents for peroxidase activity, including a colorimetricsubstrate are then added to the dish. Immobilized peroxidase, linked toREQUIEM through the primary and secondary antibodies, produces a coloredreaction product. The amount of color developed in a given time periodindicates the amount of REQUIEM protein present in the sample.Quantitative results typically are obtained by reference to a standardcurve.

A competition assay may be employed wherein antibodies specific toREQUIEM attached to a solid support and labeled REQUIEM and a samplederived from the host are passed over the solid support and the amountof label detected attached to the solid support can be correlated to aquantity of REQUIEM in the sample.

Antibodies

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler, G. and Milstein, C.,Nature 256: 495-497 (1975), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today 4: 72 (1983) andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc. (1985).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshumanized antibodies to immunogenic polypeptide products of thisinvention.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or purify the polypeptide of thepresent invention by attachment of the antibody to a solid support forisolation and/or purification by affinity chromatography.

Thus, among others, antibodies against REQUIEM may be employed toinhibit the action of such REQUIEM polypeptides, for example, in thetreatment of Alzheimer's disease, Parkinson's disease, rheumatoidarthritis, septic shock, sepsis, stroke, CNS inflammation, osteoporosis,ischemia reperfusion injury, cell death associated with cardiovasculardisease, polycystic kidney disease, apoptosis of endothelial cells incardiovascular disease, degenerative liver disease, MS, ALS, cererbellardegeneration, ischemic injury, myocardial infarction, AIDS,myelodysplastic syndromes, aplastic anemia, male pattern baldness, andhead injury damage.

REQUIEM binding molecules and assay

This invention also provides a method for identification of molecules,such as receptor molecules, that bind REQUIEM. Genes encoding proteinsthat bind REQUIEM, such as receptor proteins, can be identified bynumerous methods known to those of skill in the art, for example, ligandpanning and FACS sorting. Such methods are described in many laboratorymanuals such as, for instance, Coligan et al., Current Protocols inImmunology 1(2): Chapter 5 (1991).

For instance, expression cloning may be employed for this purpose. Tothis end polyadenylated RNA is prepared from a cell responsive toREQUIEM, a cDNA library is created from this RNA, the library is dividedinto pools and the pools are transfected individually into cells thatare not responsive to REQUIEM. The transfected cells then are exposed tolabeled REQUIEM. REQUIEM can be labeled by a variety of well-knowntechniques including standard methods of radio-iodination or inclusionof a recognition site for a site-specific protein kinase.) Followingexposure, the cells are fixed and binding of REQUIEM is determined.These procedures conveniently are carried out on glass slides.

Pools are identified of cDNA that produced REQUIEM-binding cells.Sub-pools are prepared from these positives, transfected into host cellsand screened as described above. Using an iterative sub-pooling andre-screening process, one or more single clones that encode the putativebinding molecule, such as a receptor molecule, can be isolated.

Alternatively a labeled ligand can be photoaffinity linked to a cellextract, such as a membrane or a membrane extract, prepared from cellsthat express a molecule that it binds, such as a receptor molecule.Cross-linked material is resolved by polyacrylamide gel electrophoresis("PAGE") and exposed to X-ray film. The labeled complex containing theligand-receptor can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing can be used to design unique or degenerateoligonucleotide probes to screen cDNA libraries to identify genesencoding the putative receptor molecule.

Polypeptides of the invention also can be used to assess REQUIEM bindingcapacity of REQUIEM binding molecules, such as receptor molecules, incells or in cell-free preparations.

Agonists and antagonists--assays and molecules

The invention also provides a method of screening compounds to identifythose which enhance or block the action of REQUIEM on cells, such as itsinteraction with REQUIEM-binding molecules such as receptor molecules.An agonist is a compound which increases the natural biologicalfunctions of REQUIEM or which functions in a manner similiar to REQUIEM,while antagonists decrease or eliminate such functions.

For example, a cellular compartment, such as a membrane or a preparationthereof, such as a membrane-preparation, may be prepared from a cellthat expresses a molecule that binds REQUIEM, such as a molecule of asignaling or regulatory pathway modulated by REQUIEM. The preparation isincubated with labeled REQUIEM in the absence or the presence of acandidate molecule which may be a REQUIEM agonist or antagonist. Theability of the candidate molecule to bind the binding molecule isreflected in decreased binding of the labeled ligand. Molecules whichbind gratuitously, i.e., without inducing the effects of REQUIEM onbinding the REQUIEM binding molecule, are most likely to be goodantagonists. Molecules that bind well and elicit effects that are thesame as or closely related to REQUIEM are agonists.

REQUIEM-like effects of potential agonists and antagonists may bymeasured, for instance, by determining activity of a second messengersystem following interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that ofREQUIEM or molecules that elicit the same effects as REQUIEM. Secondmessenger systems that may be useful in this regard include but are notlimited to AMP guanylate cyclase, ion channel or phosphoinositidehydrolysis second messenger systems.

Another example of an assay for REQUIEM antagonists is a competitiveassay that combines REQUIEM and a potential antagonist withmembrane-bound REQUIEM receptor molecules or recombinant REQUIEMreceptor molecules under appropriate conditions for a competitiveinhibition assay. REQUIEM can be labeled, such as by radioactivity, suchthat the number of REQUIEM molecules bound to a receptor molecule can bedetermined accurately to assess the effectiveness of the potentialantagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a receptor molecule, without inducingREQUIEM-induced activities, thereby preventing the action of REQUIEM byexcluding REQUIEM from binding.

Potential antagonists include a small molecule which binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such as receptor molecules, such thatnormal biological activity is prevented. Examples of small moleculesinclude but are not limited to small organic molecules, peptides orpeptide-like molecules. Another antagonist is an oligopeptide comprisingthe cleavage site recognition motif for REQUIEM.

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through antisense DNAor RNA or through triple-helix formation. Antisense techniques arediscussed, for example, in--Okano, J. Neurochem. 56: 560 (1991);OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRCPress, Boca Raton, Fla. (1988). Triple helix formation is discussed in,for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooneyet al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360(1991). The methods are based on binding of a polynucleotide to acomplementary DNA or RNA. For example, the 5' coding portion of apolynucleotide that encodes the mature polypeptide of the presentinvention may be used to design an antisense RNA oligonucleotide fromabout 10 to 40 base pairs in length. A DNA oligonucleotide is designedto be complementary to a region of the gene involved in transcriptionthereby preventing transcription and the production of REQUIEM. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into REQUIEM polypeptide. Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of REQUIEM.

Agonists targeted to defective cellular proliferation, including, forexample, cancer cells and solid tumor cells may be used for thetreatment of these diseases. Such targeting may be achieved via genetherapy of using antibody fusions.

Agonists may also be used to treat follicular lymphomas, carcinomasassociated with p53 mutations, autoimmune disorders, such as, forexample, SLE, immune-mediated glomeruonephritis; and hormone-dependenttumors, such as, for example, breast cancer, prostate cancer and ovarycancer; and viral infections, such as, for example, herpesviruses,poxviruses and adenoviruses.

The antagonists may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as hereinafter described.

The antagonists may be employed for instance to inhibit the action ofREQUIEM polypeptides, for example, in the treatment of Alzheimer'sdisease, Parkinson's disease, rheumatoid arthritis, septic shock,sepsis, stroke, CNS inflammation, osteoporosis, ischemia reperfusioninjury, cell death associated with cardiovascular disease, polycystickidney disease, apoptosis of endothelial cells in cardiovasculardisease, degenerative liver disease, MS, ALS, cererbellar degeneration,ischemic injury, myocardial infarction, AIDS, myelodysplastic syndromes,aplastic anemia, male pattern baldness, and head injury damage.

The antagonists may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as hereinafter described.

Compositions

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or the agonists or antagonists.Thus, the polypeptides of the present invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa polypeptide of the invention and a pharmaceutically acceptable carrieror excipient. Such carriers may include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol and combinationsthereof. The formulation should suit the mode of administration.

Kits

The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

Administration

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

The pharmaceutical compositions generally are administered in an amounteffective for treatment or prophylaxis of a specific indication orindications. In general, the compositions are administered in an amountof at least about 10 μg/kg body weight. In most cases they will beadministered in an amount not in excess of about 8 mg/kg body weight perday. Preferably, in most cases, dose is from about 10 μg/kg to about 1mg/kg body weight, daily. It will be appreciated that optimum dosagewill be determined by standard methods for each treatment modality andindication, taking into account the indication, its severity, route ofadministration, complicating conditions and the like.

Gene therapy

The REQUIEM polynucleotides, polypeptides, agonists and antagonists thatare polypeptides may be employed in accordance with the presentinvention by expression of such polypeptides in vivo, in treatmentmodalities often referred to as "gene therapy."

Thus, for example, cells from a patient may be engineered with apolynucleotide, such as a DNA or RNA, encoding a polypeptide ex vivo,and the engineered cells then can be provided to a patient to be treatedwith the polypeptide. For example, cells may be engineered ex vivo bythe use of a retroviral plasmid vector containing RNA encoding apolypeptide of the present invention. Such methods are well-known in theart and their use in the present invention will be apparent from theteachings herein.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by procedures known in the art. For example, apolynucleotide of the invention may be engineered for expression in areplication defective retroviral vector, as discussed above. Theretroviral expression construct then may be isolated and introduced intoa packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest. These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

Retroviruses from which the retroviral plasmid vectors herein abovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

Such vectors well include one or more promoters for expressing thepolypeptide. Suitable promoters which may be employed include, but arenot limited to, the retroviral LTR; the SV40 promoter; and the CMVpromoter described in Miller et al., Biotechniques 7: 980-990 (1989), orany other promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, RNA polymeraseIII, and β-actin promoters). Other viral promoters which may be employedinclude, but are not limited to, adenovirus promoters, thymidine kinase(TK) promoters, and B19 parvovirus promoters. The selection of asuitable promoter will be apparent to those skilled in the art from theteachings contained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention will be placed under the control of a suitable promoter.Suitable promoters which may be employed include, but are not limitedto, adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the CMV promoter; the respiratorysyncytial virus ("RSV") promoter; inducible promoters, such as the MMTpromoter, the metallothionein promoter; heat shock promoters; thealbumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, Y-2,Y-AM, PA12, T19-14X, VT-19-17-H2, YCRE, YCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, A., Human Gene Therapy 1: 5-14(1990). The vector may be transduced into the packaging cells throughany means known in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

The producer cell line will generate infectious retroviral vectorparticles, which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

EXAMPLES

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplification's, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

Certain terms used herein are explained in the foregoing glossary.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., cited above.

All parts or amounts set out in the following examples are by weight,unless otherwise specified.

Unless otherwise stated size separation of fragments in the examplesbelow was carried out using standard techniques of agarose andpolyacrylamide gel electrophoresis ("PAGE") in Sambrook and numerousother references such as, for instance, by Goeddel et al., Nucleic AcidsRes. 8: 4057 (1980).

Unless described otherwise, ligations were accomplished using standardbuffers, incubation temperatures and times, approximately equimolaramounts of the DNA fragments to be ligated and approximately 10 units ofT4 DNA ligase ("ligase") per 0.5 μg of DNA.

Example 1

Expression and purification of human REQUIEM using bacteria

The DNA sequence encoding human REQUIEM in the polynucleotide of thecDNA library was amplified using PCR oligonucleotide primers specific tothe amino acid carboxyl terminal sequence of the human REQUIEM proteinand to vector sequences 3' to the gene. Additional nucleotidescontaining restriction sites to facilitate cloning were added to the 5'and 3' sequences respectively.

The 5' oligonucleotide primer had the sequence 5'ATAACGAGAATTCATGGCGGCTGTGGTGGAAAATG 3' (SEQ ID NO:3) containing theunderlined EcoRI restriction site, which encodes a start AUG, followedby 1220 nucleotides of the human REQUIEM coding sequence set out in FIG.1 beginning with the first base of the first codon.

The 3' primer had the sequence 5' ACGTCTAGAGATATCCACATCAAGAGGAGTTCTGG 3'(SEQ ID NO:4) containing the underlined EcoRV restriction site followedby 20 nucleotides complementary to the last 20 nucleotides of theREQUIEM coding sequence set out in FIG. 1, including the stop codon.

The restrictions sites were convenient to restriction enzyme sites inthe bacterial expression vectors pQE-9 which were used for bacterialexpression in these examples. (Qiagen, Inc. Chatsworth, Calif. pQE-9encodes ampicillin antibiotic resistance ("Amp^(r) ") and contains abacterial origin of replication ("ori"), an IPTG inducible promoter, aribosome binding site ("RBS"), a 6-His tag and restriction enzyme sites.

The amplified human REQUIEM DNA and the vector pQE-9 both were digestedwith EcoRI and EcoRV and the digested DNAs then were ligated together.Insertion of the REQUIEM DNA into the EcoRI and EcoRV restricted vectorplaced the REQUIEM coding region downstream of and operably linked tothe vector's IPTG-inducible promoter and in-frame with an initiating AUGappropriately positioned for translation of REQUIEM.

The ligation mixture was transformed into competent E. coli cells usingstandard procedures. Such procedures are described in Sambrook et al.,cited above. E. coli strain M15/rep4, containing multiple copies of theplasmid pREP4, which expresses lac repressor and confers kanamycinresistance ("Kan^(r) "), was used in carrying out the illustrativeexample described here. This strain, which is only one of many that aresuitable for expressing REQUIEM, is available commercially from Qiagen.

Transformants were identified by their ability to grow on LB plates inthe presence of ampicillin (Amp^(r) phenotype). Plasmid DNA was isolatedfrom resistant colonies and the identity of the cloned DNA was confirmedby restriction analysis.

Clones containing the desired constructs were grown overnight ("O/N") inliquid culture in LB media supplemented with both ampicillin (100 ug/ml)and kanamycin (25 ug/ml).

The O/N culture was used to inoculate a large culture, at a dilution ofapproximately 1:100 to 1:250. The cells were grown to an optical densityat 600 mn (OD⁶⁰⁰) of between 0.4 and 0.6.Isopropyl-B-D-thiogalactopyranoside ("IPGT") was then added to a finalconcentration of 1 mM to induce transcription from lac repressorsensitive promoters, by inactivating the lacI repressor. Cellssubsequently were incubated further for 3 to 4 hours. Cells then wereharvested by centrifugation and disrupted, by standard methods.Inclusion bodies were purified from the disrupted cells using routinecollection techniques, and protein was solubilized from the inclusionbodies into 8M urea. The 8M urea solution containing the solubilizedprotein was passed over a PD-10 column in 2× phosphate buffered saline("PBS"), thereby removing the urea, exchanging the buffer and refoldingthe protein. The protein was purified by a further step ofchromatography to remove endotoxin. Then, it was sterile filtered. Thesterile filtered protein preparation was stored in 2× PBS at aconcentration of 95 micrograms per mL.

Example 2

Cloning and expression of human REQUIEM in a baculovirus expressionsystem

The cDNA sequence encoding the full length human REQUIEM protein, in thecDNA library clone is amplified using PCR oligonucleotide primerscorresponding to the 5' and 3' sequences of the gene:

The 5' primer has the sequence 5' ATAACGAGAATTCATGGCGGCTGTGGTGGAAAATG 3'(SEQ ID NO: 3) containing the underlined EcoRI restriction enzyme sitefollowed by 1220 bases of the sequence of REQUIEM of FIG. 1. Insertedinto an expression vector, as described below, the 5' end of theamplified fragment encoding human REQUIEM provides an efficient signalpeptide. An efficient signal for initiation of translation in eukaryoticcells, as described by Kozak, M., J. Mol. Biol. 196: 947-950 (1987) isappropriately located in the vector portion of the construct.

The 3' primer has the sequence 5' ACGTCTAGAGATATCCACATCAAGAGGAGTTCTGG 3'(SEQ ID NO:4) containing the underlined EcoRV restriction followed bynucleotides complementary to the last 20 nucleotides of the REQUIEMcoding sequence set out in FIG. 1, including the stop codon.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit ("Geneclean," BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with EcoRI and EcoRV and again ispurified on a 1% agarose gel. This fragment is designated herein F2.

The vector pRG1 is used to express the REQUIEM protein in thebaculovirus expression system, using standard methods, such as thosedescribed in Summers et al, A MANUAL OF METHODS FOR BACULOVIRUS VECTORSAND INSECT CELL CULTURE PROCEDURES, Texas Agricultural ExperimentalStation Bulletin No. 1555 (1987). This expression vector contains thestrong polyhedrin promoter of the Autographa californica nuclearpolyhedrosis virus (AcMNPV) followed by convenient restriction sites.The signal peptide of AcMNPV gp67, including the N-terminal methionine,is located just upstream of a EcoRI site. The polyadenylation site ofSV40 is used for efficient polyadenylation. For an easy selection ofrecombinant virus the beta-galactosidase gene from E. coli is insertedin the same orientation as the polyhedrin promoter and is followed bythe polyadenylation signal of the polyhedrin gene. The polyhedrinsequences are flanked at both sides by viral sequences for cell-mediatedhomologous recombination with wild-type viral DNA to generate viablevirus that express the cloned polynucleotide.

Many other baculovirus vectors could be used in place of pA2-GP, such aspAc373, pVL941 and pAcIM1 provided, as those of skill readily willappreciate, that construction provides appropriately located signals fortranscription, translation, trafficking and the like, such as anin-frame AUG and a signal peptide, as required. Such vectors aredescribed in Luckow et al., Virology 170: 31-39, among others.

The plasmid is digested with the restriction enzymes EcoRI and EcoRV andthen is dephosphorylated using calf intestinal phosphatase, usingroutine procedures known in the art. The DNA is then isolated from a 1%agarose gel using a commercially available kit ("Geneclean" BIO 101Inc., La Jolla, Calif.). This vector DNA is designated herein "V2".

Fragment F2 and the dephosphorylated plasmid V2 are ligated togetherwith T4 DNA ligase. E. coli HB101 cells are transformed with ligationmix and spread on culture plates. Bacteria are identified that containthe plasmid with the human REQUIEM gene by digesting DNA from individualcolonies using EcoRI and EcoRV and then analyzing the digestion productby gel electrophoresis. The sequence of the cloned fragment is confirmedby DNA sequencing. This plasmid is designated herein pBacREQUIEM.

5 μg of the plasmid pBacREQUIEM is co-transfected with 1.0 μg of acommercially available linearized baculovirus DNA ("BaculoGold™baculovirus DNA", Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmidpBacREQUIEM are mixed in a sterile well of a microtiter plate containing50 μl of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27° C. After 5 hours the transfection solution is removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. The plate is put back into an incubator and cultivation iscontinued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, cited above. An agarosegel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used toallow easy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a "plaqueassay" of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10).

Four days after serial dilution, the virus is added to the cells. Afterappropriate incubation, blue stained plaques are picked with the tip ofan Eppendorf pipette. The agar containing the recombinant viruses isthen resuspended in an Eppendorf tube containing 200 μl of Grace'smedium. The agar is removed by a brief centrifugation and thesupernatant containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. A clonecontaining properly inserted REQUIEM is identified by DNA analysisincluding restriction mapping and sequencing. This is designated hereinas V-REQUIEM.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-REQUIEM at a multiplicity of infection ("MOI") of about 2(about 1 to about 3). Six hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Gaithersburg). 42 hours later, 5 μCi of ³⁵S-methionine and 5 μCi of ³⁵ S cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then they areharvested by centrifugation, lysed and the labeled proteins arevisualized by SDS-PAGE and autoradiography.

Example 3

Expression of REQUIEM in COS cells

The expression plasmid, REQUIEM HA, is made by cloning a cDNA encodingREQUIEM into the expression vector pcDNAI/Amp (which can be obtainedfrom Invitrogen, Inc.).

The expression vector pcDNAI/amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcell; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron, and a polyadenylation signal arranged so that a cDNAconveniently can be placed under expression control of the CMV promoterand operably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker.

A DNA fragment encoding the entire REQUIEM precursor and a HA tag fusedin flame to its 3' end is cloned into the polylinker region of thevector so that recombinant protein expression is dire by the CMVpromoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein described by Wilson et al., Cell 37: 767(1984). The fusion of the HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

The plasmid construction strategy is as follows.

The REQUIEM cDNA of the clone from a cDNA library is amplified usingprimers that contained convenient restriction sites, much as describedabove regarding the construction of expression vectors for expression ofREQUIEM in E. coli and S. fugiperda.

To facilitate detection, purification and characterization of theexpressed REQUIEM, one of the primers contains a hemagglutinin tag ("HAtag") as described above.

Suitable primers include that following, which are used in this example.

The 5' primer, containing the underlined EcoRI site, an AUG start codonand 6 codons thereafter, forming the hexapeptide haemaglutinin tag, hasthe following sequence; 5' ATAACGAGAATTCATGGCGGCTGTGGTGGAAAATG 3' (SEQID NO:3).

The 3' primer, containing the underlined EcoRV site and 20 bp of 3'coding sequence (at the 3' end) has the following sequence; 5'ACGTCTAGAGATATCCACATCAAGAGGAGTTCTGG 3' (SEQ ID NO:4).

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith and then ligated. The ligation mixture is transformed into E. colistrain SURE (available from Stratagene Cloning Systems, 11099 NorthTorrey Pines Road, La Jolla, Calif. 92037) the transformed culture isplated on ampicillin media plates which then are incubated to allowgrowth of ampicillin resistant colonies. Plasmid DNA is isolated fromresistant colonies and examined by restriction analysis and gel sizingfor the presence of the REQUIEM-encoding fragment.

For expression of recombinant REQUIEM, COS cells are transfected with anexpression vector, as described above, using DEAE-DEXTRAN, as described,for instance, in Sambrook et al., cited above.

Cells are incubated under conditions for expression of REQUIEM by thevector.

Expression of the REQUIEM HA fusion protein is detected byradiolabelling and immunoprecipitation, using methods described in, forexample Harlow et al., ANTIBODIES: A LABORATORY MANUAL, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To thisend, two days after transfection, the cells are labeled by incubation inmedia containing ³⁵ S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and the lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5%DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above.Proteins are precipitated from the cell lysate and from the culturemedia using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE gels and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 4

Gene therapeutic expression of human REQUIEM

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature overnight. After 24 hours at room temperature, the flask isinverted--the chunks of tissue remain fixed to the bottom of theflask--and fresh media is added (e.g., Ham's F12 media, with 10% FBS,penicillin and streptomycin). The tissue is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerges. The monolayer istrypsinized and scaled into larger flasks.

A vector for gene therapy is digested with restriction enzymes forcloning a fragment to be expressed. The digested vector is treated withcalf intestinal phosphatase to prevent self-ligation. Thedephosphorylated, linear vector is fractionated on an agarose gel andpurified.

REQUIEM cDNA capable of expressing active REQUIEM, is isolated. The endsof the fragment are modified, if necessary, for cloning into the vector.For instance, 5' overhanging may be treated with DNA polymerase tocreate blunt ends. 3' overhanging ends may be removed using S1 nuclease.Linkers may be ligated to blunt ends with T4 DNA ligase.

Equal quantities of the Moloney murine leukemia virus linear backboneand the REQUIEM fragment are mixed together and joined using T4 DNAligase. The ligation mixture is used to transform E. coli and thebacteria are then plated onto agar-containing kanamycin. Kanamycinphenotype and restriction analysis confirm that the vector has theproperly inserted gene.

Packaging cells are grown in tissue culture to confluent density inDulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS),penicillin and streptomycin. The vector containing the REQUIEM gene isintroduced into the packaging cells by standard techniques. Infectiousviral particles containing the REQUIEM gene are collected from thepackaging cells, which now are called producer cells.

Fresh media is added to the producer cells, and after an appropriateincubation period media is harvested from the plates of confluentproducer cells. The media, containing the infectious viral particles, isfiltered through a Millipore filter to remove detached producer cells.The filtered media then is used to infect fibroblast cells. Media isremoved from a sub-confluent plate of fibroblasts and quickly replacedwith the filtered media. Polybrene (Aldrich) may be included in themedia to facilitate transduction. After appropriate incubation, themedia is removed and replaced with fresh media. If the titer of virus ishigh, then virtually all fibroblasts will be infected and no selectionis required. If the titer is low, then it is necessary to use aretroviral vector that has a selectable marker, such as neo or his, toselect out transduced cells for expansion.

Engineered fibroblasts then may be injected into rats, either alone orafter having been grown to confluence on microcarrier beads, such ascytodex 3 beads. The injected fibroblasts produce REQUIEM product, andthe biological actions of the protein are conveyed to the host.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 4    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1556 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - GGCACGAGGG AACAGGGAAG ATGGCGGCTG TGGTGGAAAA TGTAGTGAAG CT - #CCTTGGGG      60    - AGCAGTACTA CAAAGATGCC ATGGAGCAGT GCCACAATTA CAATGCTCGC CT - #CTGTGCTG     120    - AGCGCAGCGT GCGCCTGCCT TTCTTGGACT CACAGACCGG AGTAGCCCAG AG - #CAATTGTT     180    - ACATCTGGAT GGAAAAGCGA CACCGGGGTC CAGGATTGGC CTCCGGACAG CT - #GTACTCCT     240    - ACCCTGCCCG GCGCTGGCGG AAAAAGCGGC GAGCCCATCC CCCTGAGGAT CC - #ACGACTTT     300    - CCTTCCCATC TATTAAGCCA GACACAGACC AGACCCTGAA GAAGGAGGGG CT - #GATCTCTC     360    - AGGATGGCAG TAGTTTAGAG GCTCTGTTGC GCACTGACCC CCTGGAGAAG CG - #AGGTGCCC     420    - CGGATCCCCG AGTTGATGAT GACAGCCTGG GCGAGTTTCC TGTGACCAAC AG - #TCGAGCGC     480    - GAAAGCGGAT CCTAGAACCA GATGACTTCC TGGATGACCT CGATGATGAA GA - #CTATGAAG     540    - AAGATACTCC CAAGCGTCGG GGAAAGGGGA AATCCAAGGG TAAGGGTGTG GG - #CAGTGCCC     600    - GTAAGAAGCT GGATGCTTCC ATCCTGGAGG ACCGGGATAA GCCCTATGCC TG - #TGACAATA     660    - GTTTCAAACA AAAGCATACC TCGAAAGCGC CCCAGAGAGT TTGTGGAAAA CG - #TTACAAGA     720    - ACCGACCAGG CCTCAGTTAC CACTATGCCC ACTCCCACTT GGCTGAGGAG GA - #GGGCGAGG     780    - ACAAGGAAGA CTCTCAACCA CCCACTCCTG TTTCCCAGAG GTCTGAGGAG CA - #GAAATCCA     840    - AAAAGGGTCC TGATGGATTG GCCTTGCCCA ACAACTACTG TGACTTCTGC CT - #GGGGGACT     900    - CAAAGATTAA CAAGAAGACG GGACAACCCG AGGAGCTGGT GTCCTGTTCT GA - #CTGTGGCC     960    - GCTCAGGGCA TCCATCTTGC CTCCAATTTA CCCCCGTGAT GATGGCGGCA GT - #GAAGACAT    1020    - ACCGCTGGCA GTGCATCGAG TGCAAATGTT GCAATATCTG CGGCACCTCC GA - #GAATGACG    1080    - ACCAGTTGCT CTTCTGTGAT GACTGCGATC GTGGCTACCA CATGTACTGT CT - #CACCCCGT    1140    - CCATGTCTGA GCCCCCTGAA GGAAGTTGGA GCTGCCACCT GTGTCTGGAC CT - #GTTGAAAG    1200    - AGAAAGCTTC CATCTACCAG AACCAGAACT CCTCTTGATG TGGCCACCCA CC - #TGCTCCCC    1260    - GACATATCTA AGGCTGTTTC TCTCCTCCAC TTCATATTTC ATACCCATCT TT - #CCCTTCTT    1320    - CCTCCTCTCC TTCACAAATC CAGAGAACCT TGGGGTGGTT GTGCCAGCCT GC - #CTTTGGCA    1380    - GCTGCAAGCT GAGGTGGCAG CTCTGACCAC CTCTGGCCCC AGGCCCTCCA GG - #GAGAAAGG    1440    - AGCAACACAC TGCCCCTAGG CGTGCGTGTG GCCCAGTTTC TCTCTGCTCT CC - #ATTAAGTG    1500    - CATTCACTCT GCTTGCCTTG GGCCCAGCCC CCTGGTTGAT TACAGGGTTC AA - #GGGA    1556    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 405 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - Met Ala Ala Val Val Glu Asn Val Val Lys Le - #u Leu Gly Glu Gln Tyr    #                15    - Tyr Lys Asp Ala Met Glu Gln Cys His Asn Ty - #r Asn Ala Arg Leu Cys    #            30    - Ala Glu Arg Ser Val Arg Leu Pro Phe Leu As - #p Ser Gln Thr Gly Val    #        45    - Ala Gln Ser Asn Cys Tyr Ile Trp Met Glu Ly - #s Arg His Arg Gly Pro    #    60    - Gly Leu Ala Ser Gly Gln Leu Tyr Ser Tyr Pr - #o Ala Arg Arg Trp Arg    #80    - Lys Lys Arg Arg Ala His Pro Pro Glu Asp Pr - #o Arg Leu Ser Phe Pro    #                95    - Ser Ile Lys Pro Asp Thr Asp Gln Thr Leu Ly - #s Lys Glu Gly Leu Ile    #           110    - Ser Gln Asp Gly Ser Ser Leu Glu Ala Leu Le - #u Arg Thr Asp Pro Leu    #       125    - Glu Lys Arg Gly Ala Pro Asp Pro Arg Val As - #p Asp Asp Ser Leu Gly    #   140    - Glu Phe Pro Val Thr Asn Ser Arg Ala Arg Ly - #s Arg Ile Leu Glu Pro    145                 1 - #50                 1 - #55                 1 -    #60    - Asp Asp Phe Leu Asp Asp Leu Asp Asp Glu As - #p Tyr Glu Glu Asp Thr    #               175    - Pro Lys Arg Arg Gly Lys Gly Lys Ser Lys Gl - #y Lys Gly Val Gly Ser    #           190    - Ala Arg Lys Lys Leu Asp Ala Ser Ile Leu Gl - #u Asp Arg Asp Lys Pro    #       205    - Tyr Ala Cys Asp Asn Ser Phe Lys Gln Lys Hi - #s Thr Ser Lys Ala Pro    #   220    - Gln Arg Val Cys Gly Lys Arg Tyr Lys Asn Ar - #g Pro Gly Leu Ser Tyr    225                 2 - #30                 2 - #35                 2 -    #40    - His Tyr Ala His Ser His Leu Ala Glu Glu Gl - #u Gly Glu Asp Lys Glu    #               255    - Asp Ser Gln Pro Pro Thr Pro Val Ser Gln Ar - #g Ser Glu Glu Gln Lys    #           270    - Ser Lys Lys Gly Pro Asp Gly Leu Ala Leu Pr - #o Asn Asn Tyr Cys Asp    #       285    - Phe Cys Leu Gly Asp Ser Lys Ile Asn Lys Ly - #s Thr Gly Gln Pro Glu    #   300    - Glu Leu Val Ser Cys Ser Asp Cys Gly Arg Se - #r Gly His Pro Ser Cys    305                 3 - #10                 3 - #15                 3 -    #20    - Leu Gln Phe Thr Pro Val Met Met Ala Ala Va - #l Lys Thr Tyr Arg Trp    #               335    - Gln Cys Ile Glu Cys Lys Cys Cys Asn Ile Cy - #s Gly Thr Ser Glu Asn    #           350    - Asp Asp Gln Leu Leu Phe Cys Asp Asp Cys As - #p Arg Gly Tyr His Met    #       365    - Tyr Cys Leu Thr Pro Ser Met Ser Glu Pro Pr - #o Glu Gly Ser Trp Ser    #   380    - Cys His Leu Cys Leu Asp Leu Leu Lys Glu Ly - #s Ala Ser Ile Tyr Gln    385                 3 - #90                 3 - #95                 4 -    #00    - Asn Gln Asn Ser Ser                    405    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 35 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #       35         GCGG CTGTGGTGGA AAATG    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 35 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    #       35         ACAT CAAGAGGAGT TCTGG    __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide sequence encoding a polypeptide comprising amino acids 1to 405 of SEQ ID NO:2.
 2. The isolated polynucleotide of claim 1comprising nucleotides 1 to 1555 set forth in SEQ ID NO:1.
 3. Theisolated polynucleotide of claim 1 comprising nucleotides 21 to 12 38set forth in SEQ ID NO:1.
 4. An expression vector comprising theisolated polynucleotide of claim
 1. 5. A host cell comprising the vectorof claim
 4. 6. A process for producing a polypeptidecomprising:expressing from the host cell of claim 5 a polypeptideencoded by said polynucleotide.
 7. A process for producing a cell whichexpresses a polypeptide comprising transforming or transfecting the cellwith the vector of claim 4 such that the cell expresses the polypeptideencoded by the polynucleotide contained in the vector.
 8. The isolatedpolynucleotide of claim 1 wherein said polynucleotide is an RNAtranscript of the entire length of SEQ ID NO:1.
 9. The isolatedpolynucleotide of claim 1 wherein said polynucleotide is an RNAtranscript of the entire coding region of SEQ ID NO:1.
 10. An isolatedpolynucleotide comprising a polynucleotide sequence encoding a naturallyoccurring human variant of the polypeptide having the amino acidsequence set forth in SEQ ID NO:2.
 11. An isolated polynucleotide whichis complementary to any one of the isolated polynucleotides of claims 1,2, 3, and 8-10.
 12. The isolated polynucleotide of any one of claims 1,2, 3, and 10 wherein the polynucleotide is DNA or RNA.
 13. A process fordiagnosing a disease or a susceptibility to a disease related toexpression of the polypeptide having the amino acid sequence set forthin SEQ ID NO:2 comprising:determining a mutation in the nucleic acidsequence encoding said polypeptide by detecting an alteration in theamino acid sequence of the polypeptide encoded by said nucleic acidsequence.